CN101784884B

CN101784884B - Method and system for characterizing a pigmented biological tissue

Info

Publication number: CN101784884B
Application number: CN2008800183791A
Authority: CN
Inventors: 佐澜·克洛维可; 尼克拉·莫伊斯; 格温达尔·拉托彻; 耶维斯·苟拉斯
Original assignee: Force-A Inc; Universite Paris Sud Paris 11
Current assignee: Force-A Inc; Universite Paris Sud Paris 11
Priority date: 2007-06-01
Filing date: 2008-05-27
Publication date: 2012-02-15
Anticipated expiration: 2028-05-27
Also published as: WO2008152292A4; AU2008263711A1; WO2008152292A8; EP2179270B1; US20100184117A1; DE602008005753D1; WO2008152292A9; CA2688440A1; ES2363925T3; FR2916848A1; WO2008152292A1; FR2916848B1; AU2008263711B2; CN101784884A; ATE503178T1; EP2179270A1; AU2008263711C1

Abstract

Method for determining the content of a chromophoric and nonfluorescent compound, referred to as first compound, of a biological tissue (30) of a biological entity, wherein said biological tissue (30) also comprises a chromophoric and fluorescent compound, referred to as second compound, and wherein said method comprises at least one iteration of the following operations: emission, in the direction of said tissue (30), of a first, ''reference'', optical radiation and of a second, ''measuring'', optical radiation, each chosen so as to induce a fluorescence radiation from said second compound, wherein each of said first and second radiations is partially absorbed by said first compound, measurement of said fluorescence radiations induced by said first and second radiations, and determination, from said measurement, of the content of said first compound in said tissue; characterized in that said method also comprises at least one compensation for saturation of the measurement due to too great an absorption of the measuring radiation by said first compound, wherein said compensation comprises a choice, for said measuring radiation, of a wavelength corresponding to a weaker absorption in the absorption spectrum of the first compound.

Description

Method and system for characterizing pigmented biological tissue

The present invention relates to a method for characterizing pigmented biological tissue. The present aspect also relates to a system for performing the method.

More specifically, the present invention relates to methods and systems for characterizing the tissue of a biological entity (e.g., berry) that includes a first chromogenic compound (e.g., anthocyanin or brassinol) that is only slightly or not fluorescent and a second fluorescent chromogenic compound (e.g., chlorophyll). The characterization is aimed at determining the content of the first compound in the biological tissue. This characterization of biological tissues is very important. For example, in the wine making field, the content of anthocyanins in grapes represents information related to their maturity and the quality of the wine brewed from the grapes. In the nutritional field, the content of brassinol in the epidermis of fruits and vegetables is an indicator of their nutritional value.

Document FR 2830325 discloses an optical instrument capable of measuring the light absorption characteristics of a biological tissue sample comprising a first non-fluorescent chromophoric compound and a second fluorescent chromophoric compound by means of a screening effect. The method disclosed in this document comprises measuring fluorescence generated by exciting a second compound by irradiating a biological tissue sample with first and second radiations having different wavelengths. The so-called reference radiation is slightly or not absorbed at all by the first compound, while the so-called measurement radiation is absorbed relatively well. By measuring the fluorescence generated by the two radiations and obtaining the ratio of the measured fluorescence, the method can measure the light absorption characteristics of the sample and thus the content of the first compound in the biological tissue.

However, the development of the first non-fluorescent chromonic compound (concentration) saturates the measurement of fluorescent radiation produced by the first radiation. When the concentration of the first compound in the tissue increases, the absorption of the measurement radiation by the first compound also increases. The measurement radiation generates increasingly weaker fluorescence radiation and can no longer be distinguished from the measurement noise. The reference radiation may also be affected when the concentration of the first compound in the tissue increases. In the present invention, these effects are referred to as "saturation".

Most of the methods and systems currently known are limited to characterizing biological tissue before saturation is reached. These systems, including those based on colorimetry, not only suffer from saturation problems, but they also suffer from problems associated with deposits that may appear on biological tissue and may affect the measurements. It is also possible to document systems based on infrared spectroscopy which suffer from problems associated with the presence of water in biological tissues and determine the content in the first compound based on empirical chemometric inferences.

Other methods and systems for characterizing biological tissue avoid saturation by dilution operations in the laboratory. A drawback of these methods and systems is their destructive nature.

The aim of the present invention is to propose a novel method and system allowing to supersaturate biological tissues in a non-destructive and non-invasive manner.

It is another object of the present invention to propose a new method and system allowing to perform in situ supersaturated characterisation of biological tissues.

The invention therefore proposes a method for determining the content of a non-fluorescent chromophoric compound, referred to as first compound, in a tissue of a biological entity, said biological tissue also comprising a fluorescent chromophoric compound, referred to as second compound, said method comprising at least one iteration of the following operations:

-emitting, by an emitting device, a first radiation, called measurement optical radiation, and a second radiation, called reference optical radiation, in the direction of the tissue, each of the first and second radiation being selected to generate a fluorescent radiation of the second compound, each of the first and second radiation being partially absorbed by the first compound;

-measuring the fluorescence radiation induced by the first and second radiation by a measuring device; and

-determining the content of the first compound in the tissue from the first measurement.

The method according to the invention is characterized in that it further comprises at least one compensation of the measurement saturation, which occurs due to too high absorption of the measurement radiation by the first compound, the compensation comprising: selecting a wavelength for the measurement radiation that corresponds to a lower absorption in the absorption spectrum of the first compound.

Thus, by shifting the wavelength of the measurement radiation towards a wavelength of lower absorption in the absorption spectrum of the first compound, the method according to the invention may perform the characterization of the biological tissue of the biological entity when the supersaturation is caused by an excessively high content of the first compound (i.e. an excessively high absorption of the measurement radiation by the first compound). Furthermore, since there is no need to operate in a laboratory, the method according to the invention is non-destructive and non-invasive and can be performed in situ.

Advantageously, the method according to the invention further comprises emitting, by the emitting means, a third optical radiation, called complementary optical radiation, in the direction of said biological tissue, said third optical radiation being selected to cause a fluorescence radiation of said second compound. In this case, the method according to the invention comprises measuring said fluorescent radiation induced by said supplementary radiation. This measurement is used to determine whether the reference radiation is affected by the first compound. In fact, from the ratio of the fluorescence radiation induced by the two radiations, reference radiation and supplementary radiation, and the variation of this ratio, it can be determined whether the reference radiation is affected by the first compound. When it is detected that the reference radiation is affected by the first compound, a saturation compensation has to be performed, for example by inverting the measurement ratio.

According to a first aspect of the invention, the wavelength selected for the measuring radiation after saturation corresponds to a wavelength in the vicinity of the wavelength of the reference radiation before saturation, when the content of the first compound in the tissue is increased such that the first compound influences the reference radiation. According to a first aspect of the invention, a new wavelength is selected for the reference radiation when saturation occurs. The new wavelength selected for measurement radiation may be the wavelength of the reference radiation before saturation. When the distance between the biological tissue and the emitting means and the measuring means is fixed, reference radiation does not have to be used. However, even at saturation, the first measurement can be used in measuring the ratio.

When the distance between the biological tissue and the emitting means and the measuring means is not fixed, the new wavelength selected for the reference radiation after saturation may correspond to a wavelength that is only slightly affected or not affected at all by the content of the first compound. More particularly, the wavelength of the reference radiation after saturation may correspond to the wavelength of the supplementary radiation.

According to a second aspect of the invention, when the reference wavelength is not influenced by the content of the first compound in the tissue, the wavelength selected for the measuring radiation corresponds to a smaller absorption of the first compound than a limiting wavelength for which a potential saturation would occur with a desired maximum content of the first compound in the biological tissue. In the series of measurements, the potential maximum content of the first compound may be determined. From this potential maximum and the absorption spectrum of the first compound, the maximum absorption that can be obtained can be calculated. A limiting wavelength of the measuring radiation is determined, which corresponds to the saturation of the maximum absorption. Thus, if the wavelength selected for the measuring radiation corresponds to a smaller absorption than the limiting wavelength, no saturation of the measuring radiation occurs in the measuring series. The limiting wavelength may be determined from the saturation peak. In this case, the measurement wavelength is selected relative to the saturation peak. Thus, the method according to the invention may comprise determining a shift from the saturation peak to avoid saturation or to obtain a content value of the first compound that develops in a more linear manner.

According to the third aspect of the present invention, when the reference wavelength is not affected by the content of the first compound in the tissue, the saturation compensation may be performed when the first compound in the tissue causes the measurement to be saturated, the compensation being repeatedly performed at each saturation. After performing a measurement series with measurement radiation having a first wavelength, for example corresponding to a saturation peak, when a first saturation is reached, the wavelength of the measurement radiation may be shifted towards a second wavelength corresponding to a lower absorption than the first wavelength, so that the second wavelength does not produce a measurement saturation. A second series of measurements may then be taken until a new saturation is reached. The wavelength of the measurement radiation may then be shifted towards a third wavelength corresponding to a lower absorption than the second wavelength, and so on. After selecting a new wavelength, the absorption difference can be taken into account to calculate the content of the first compound in the tissue. The absorption difference may be obtained by studying an absorption spectrum of the first compound, which provides calibration values for the different selected wavelengths.

Advantageously, the three aspects of the invention described above can be combined.

Advantageously, the method according to the invention may comprise modifying the wavelength of said measurement radiation and/or of said reference radiation according to a physicochemical characteristic having an effect on the absorption spectrum of said first compound and/or on the fluorescence spectrum of said second compound.

These physicochemical characteristics are those selected from the following list:

-the pH of the biological tissue;

-a secondary colour effect in the biological tissue affecting the first compound; and

-an effect of at least one change in the support structure of the second compound in the biological tissue.

The wavelength of the measuring radiation and/or the reference radiation can also be modified, the absorption spectrum of the first compound or the fluorescence spectrum of the second compound, depending on the presence of new compounds in the biological tissue (e.g. by covalent changes).

Advantageously, the measurement radiation (or reference radiation) may be emitted at a predetermined intensity, while the reference radiation (or measurement radiation) is emitted at a variable intensity, such that the fluorescence radiation induced by each of said reference radiation and said measurement radiation has an equal intensity.

The intensity of the first radiation may be adjusted in dependence on a control signal, which is related to the fluorescence radiation induced by the measurement radiation and the reference radiation.

Furthermore, the intensities of the measurement radiation and the reference radiation may be varied alternately by phase shifting at a predetermined frequency, which may be 1kHz, for example.

Advantageously, the method according to the invention may comprise synchronizing the radiation emitted by the emitting means by phase modulation.

Each measurement radiation may be emitted in pulses.

Determining the amount of the first compound may comprise calculating a ratio of (fluorescence induced by the reference radiation) divided by (fluorescence induced by the measurement radiation).

According to an advantageous feature of the method of the invention, the measurement of said fluorescence radiation:

-performed on a tissue sample taken from a biological entity;

or directly on the biological entity in a non-destructive and non-invasive manner, i.e. without taking a sample.

The method according to the invention may advantageously comprise determining the development over time of the content of said first compound in said biological tissue. The progression may be determined in terms of time units (which may be days). For example, a graphical representation showing the development of compound content over a selected time unit may be presented.

In a non-limiting embodiment of the first aspect of the method according to the present invention, the biological tissue is grape peel; the first compound is an anthocyanin; the second compound is chlorophyll. When the distance between the tissue and the emitting and measuring devices is fixed:

-the wavelength of the measuring radiation before saturation is 530 nm;

-the wavelength of the reference radiation before saturation is 650 nm;

-the wavelength of the supplementary radiation is 450 nm;

-the wavelength of the measuring radiation after saturation is 650 nm;

no reference radiation after saturation.

Still in the case of this embodiment, when the distance between the tissue and the emitting and measuring devices is not fixed:

-the wavelength of the measuring radiation before saturation is 530 nm;

-the wavelength of the reference radiation before saturation is 650 nm;

-the wavelength of the supplementary radiation is 450 nm;

-the wavelength of the measuring radiation after saturation is 650 nm;

the wavelength of the reference radiation after saturation is 450 nm.

In this case, the content of anthocyanin in the grape peel can be determined according to the following relationship:

ANTH_{before saturation}＝logFER_ANTH_GREEN-logFER_CHL_G

ANTH_{After saturation}＝C+logFER_ANTH_RED-logFER_CHL_R

Wherein,

1.logFER_ANTH_GREEN＝log(FRF_{red wine}/FRF_Green)

2.logFER_ANTH_RED＝log(FRF_{Blue (B)}/FRF_{Red wine})

3.logFER_CHL_G＝log(FRF_{Red wine}/FRF_Green)

4.logFER_CHL_R＝log(FRF_{Blue (B)}/FRF_{Red wine})

In the context of these expressions,

●FRF_{red wine}Fluorescence radiation corresponding to chlorophyll induced by radiation emitted at 650nm (i.e. reference radiation before saturation and measurement radiation after saturation);

●FRF_greenFluorescence radiation corresponding to chlorophyll induced by radiation emitted at 530nm (i.e. measurement radiation before saturation);

●FRF_{blue (B)}Fluorescence radiation corresponding to chlorophyll induced by radiation emitted at 450nm (i.e., supplementary radiation before saturation and reference radiation after saturation);

● expressions 3 and 4 correspond to at least one measurement performed before anthocyanin appears in the grape peel. Used as a reference for measurements performed after the appearance of anthocyanin; and

● C is ANTH at saturation time_{After saturation}Equal to ANTH_{Before saturation}Is constant.

In another non-limiting embodiment according to the third aspect of the present invention, the biological tissue is grape peel; the first compound is a flavonol; the second compound is chlorophyll:

-the wavelength of the measuring radiation before saturation is about 375 nm;

-the wavelength of said reference radiation before saturation is about 650 nm;

-the wavelength of the measuring radiation after saturation is about 450 nm; and

the wavelength of the reference radiation after saturation remains about 650 nm.

According to another aspect of the invention, a system for implementing the method according to the invention is proposed.

Other features and advantages of the present invention will be apparent from the following drawings and detailed description of non-limiting embodiments.

FIG. 1 is a diagram of a first embodiment of a system according to the present invention;

FIG. 2 is a diagram of a second embodiment of a system according to the present invention;

fig. 3 is a diagram of the measurement principle in a first embodiment according to the present invention;

fig. 4 is a diagram of the measurement principle in a second embodiment according to the present invention;

FIGS. 5, 6 and 7 are diagrams of a system according to a second embodiment of the invention;

fig. 8 is a representation of chlorophyll fluorescence spectra, anthocyanin absorption spectra, brass alcohol absorption spectra, and the different radiations and filters used according to the first aspect of the method of the invention;

fig. 9 is a representation of the results obtained when measuring anthocyanin content in grape skins in accordance with the first aspect of the method of the invention;

FIG. 10 is a representation of chlorophyll fluorescence spectra, anthocyanin absorption spectra, and the different radiations and filters used in accordance with the second aspect of the method of the invention;

FIG. 11 is a representation of the results obtained when measuring anthocyanin content in grape peel in accordance with the second aspect of the method of the invention;

FIG. 12 is a representation of the results obtained when measuring anthocyanin content in grape peel in accordance with the combination of the first and second aspects of the method of the invention;

fig. 13 is an example representation of the variation of the absorbance spectrum of anthocyanins as a function of pH.

The non-limiting embodiments to be described below relate to a system for measuring the content of a first non-fluorescent chromophoric compound belonging to the family of polyphenols in a tissue of a biological entity, which also comprises a fluorescent chromophoric compound that is chlorophyll. The measurement of polyphenols is carried out by chlorophyll fluorescence as described in document FR 2830325.

Fig. 1 and 2 are illustrations of two embodiments of a system according to the present invention. In both embodiments, the system takes the form of two parts: a transmitting part 10 and a receiving part 20. In the first embodiment shown in fig. 1, the emitting portion 10 and the receiving portion 20 are respectively disposed on both sides of the plant tissue sample 30, whereas in the second embodiment shown in fig. 2, the emitting portion 10 and the receiving portion 20 are disposed on the same side of the biological tissue sample 30.

In either embodiment, the emitting portion 10 includes three

radiation sources

11, 12, and 13 that irradiate the front surface of the biological sample 30. As shown in fig. 1 and 2, each of the sources 11 to 13 is associated with one of the pulsed power supplies 14 to 16 and is supplied by a synchronization signal S_SAnd (5) controlling. Sources 11 to 13 are used to initiate fluorescence of chlorophyll. The emitting portion 10 may further comprise an optical filter (not shown) between the sources 11 to 13 and the sample 30 to filter undesired components in the radiation 11 to 13 emitted towards the sample 30.

The receiving part 20 of the system according to the invention comprises a detector 21 associated with an optical filter F2, i.e. a silicon photodiode providing an electrical signal depending on the detected fluorescent radiation, the optical filter F2 being located between the sample 30 and the detector 21. The function of the filter F2 is:

preventing radiation from the excitation sources 11 to 13 from passing through the detector 21;

partially or completely transmit chlorophyll fluorescence emission induced by the optical radiation emitted by sources 11 to 13.

The receiving part 20 of the system according to the invention also comprises a component 22 comprising control and calculation means, accessible on the one hand by means of the synchronization signal S_sThe synchronization of the power supplies 14 to 14 is performed and can be based on the measurement signal S provided by the detector 21_mDetermines the amount of the first compound in the tissue sample 30.

Fig. 3 and 4 are diagrams of the measurement principle and the optical path of different radiations in the first and second embodiments, respectively. Referring to fig. 3 and 4, the sources 11 to 13 are preferably Light Emitting Diodes (LEDs) and illuminate the same face 31 of the sample 30. Sources 11 to 13 emit three

radiations

111, 121 and 131, respectively, intended to provoke the fluorescence of chlorophyll 33 and three

fluorescence radiations

112, 122 and 132, respectively. In a first embodiment, the receiving section 20 of the system is located on the opposite side of the leaf or berry with respect to the emitting section 10, the fluorescent radiation emitted by the chlorophyll being directed towards the side opposite to the sources 11 to 13 and detected by the detector 21. In fact, in the first embodiment, the detector 21 is used to detect the fluorescence emitted towards the rear surface 35 of the sample to measure the content of polyphenols 34.

Fig. 4 shows

fluorescence radiation

112, 122 and 132, which is induced by

radiation

111, 121 and 131 and emitted by chlorophyll towards

sources

11, 12 and 13. In the second embodiment, the receiving part 20 and the emitting part 10 of the system are located on the same side of the leaf or berry, the fluorescent radiation emitted by the chlorophyll is directed towards the sources 11 to 13 and detected by the detector 21. In fact, in the second embodiment, the detector 21 is used to detect the fluorescence emitted towards the front surface 31 of the leaf to measure the content of polyphenols 34.

In either embodiment, the wavelength of each of the sources 11 to 13 is selected according to the absorption band of the compound to be measured, its technical characteristics (e.g. spectral purity and power), its commercial availability and cost, and the commercial availability and cost of the filter F2 associated with the detector 21.

When the fluorescence is isotropic, the first and second embodiments are substantially equivalent. The second embodiment is the preferred embodiment and the system according to the invention can be used both in situ and remotely from the biological tissue to be characterized. Furthermore, in a second embodiment, the system according to the invention can be applied directly to a biological entity in a non-destructive and non-invasive manner, i.e. without taking a sample of biological tissue.

Fig. 5 to 7 show different views of a device 50 manufactured according to a second embodiment of the system according to the invention, which is a preferred embodiment of the system according to the invention. Fig. 5 shows a front cross-sectional view of the device 50, fig. 6 shows an isometric view thereof, and fig. 7 shows another cross-sectional view from the side. The apparatus 50 allows direct measurements on a grape string 60 comprising a plurality of berries 70.

Will be referred to as

Embodiments of (1) are based on a portable cartridge 510 powered by a battery 5121, the battery 5121 may be remote from or integrated into the handle, the cartridge 510 having a measurement surface 514 and a user interface including a screen 5152 and control elements such as buttons or

keys

5101 and 5102. The cartridge may be supported by components forming a handle 512, the handle 512 including a connector for a replaceable battery 5121 or a remotely portable battery.

The cartridge 510 further comprises a cylindrical member 513 extending towards the side opposite to the interface and carrying a measuring surface at its end. The measuring surface 514 is surrounded by a guard 5130, which guard 5130 is more or less opaque and possibly detachable, so that interference from ambient light can be reduced and a reference point with respect to the measuring surface 514 is provided in terms of optical measuring distances.

The measurement surface 514 includes a set of detectors 540 that cover the fluorescence wavelengths to be measured. In the embodiment described herein, the detector set 540 includes three

detectors

541, 542, and 543, which are adjacent to each other and collectively converge in an equilateral triangle at the center of the measurement surface 514. The three detectors are oriented parallel to each other about the detection axis 5140, or slightly convergent about the detection axis 5140. Each of the

detectors

541, 542, and 543 includes a detection element (here, a silicon photodiode 5420 of approximately 2cm x 2 cm) and detects light in the determined blue-green, red, and far-red wavebands, respectively. The detection bandwidth is obtained by means of a color filter or a high-pass filter and an interference filter. The combination of these two types of filters allows for better filtering, which is especially required to prevent the detector from receiving the radiation emitted by the excitation source.

It should be noted that the detector receives the fluorescence light to be measured directly, without using optics for convergence, convergence or collimation. Only a single detection element, the photodiode 5420 (fig. 7), is required for each detector, which is chosen to be large enough to obtain good sensitivity, so that the collection optics can be omitted. In this manner, the detection elements may receive radiation 549 emanating from all of target region 591 illuminated by the excitation emitter.

This configuration allows for the use of relatively small detection elements and eliminates the need for converging optics. In addition to saving the cost of optics, size requirements, adjustments and depth of field constraints are avoided.

The measurement surface 514 has a concave conical peripheral surface supporting several sets of emitters which may emit excitation light of different wavelengths and which are distributed in a circle around the set of detectors 540.

These emitters comprise an ultraviolet emitter group 520 comprising six UV emitters 521 to 526, distributed on a circle around the detector group 540 as a group of two adjacent emitters, three groups of emitters each spaced by 120 °.

Each of these sources comprises a source (here a uv LED 527) located in a parabolic reflector 5281, forming a beam of light of about 30 °. The reflector is mounted on a base 5282, and the base 5282 determines the position of its beam relative to the detection axis 5140. Optionally, the UV emitter may also use refractive and catadioptric devices to improve the convergence of the emitted beam.

The emitters also comprise a set 530 of visible light emitters, comprising three

emitters

531, 532 and 533, distributed around the set 540 on the same circle as the set 520 of UV emitters, at 120 ° intervals and interposed between the UV emitters. Each visible light emitter comprises a source comprising an array of spaced apart red, green and blue LEDs, integrated in a common assembly 534 measuring approximately 4.5cm on a side and having a power of 3 x 15W, and covered by a transparent plastic sheet forming an array of converging microlenses. The common component 534 is mounted on a block 536 forming a radiator, the shape of which determines the orientation of the source relative to the detection axis 5140. The emitter also includes a wide-passband bandpass filter so that emission in the wavelengths used for fluorescence detection, especially towards far-red, can be limited.

As an alternative to the RGB (red-green-blue) sources described herein, a monochromatic excitation source may be used, for example an amber source in the form of a high power monochromatic LED array continuously emitting power in the order of 200W.

The excitation emitter is oriented to achieve uniform illumination of the target region 591 even in the case of heterogeneous objects and/or three-dimensions.

In embodiments of short-range applications, for example using UV excitation, the emitter's beam is directed to converge towards axes a541, a542, and a543 of

detectors

541, 542, and 543. More specifically, the axes a541, a542, and a543 of the excitation beam intersect each other and converge at the same point P5140 at the optimal measurement distance with the detection axis 5140. In the depicted embodiment, the convergence point P5140 is located 10 to 20cm, such as about 15cm, from the detector set 540.

The emitted light beams do not converge and show some opening or divergence may limit the constraints that affect the measured distance. In fact, when the target (here the bunch 60) is within beams 529 and 539 of the emitter, it is uniformly illuminated towards different surfaces of the detector set 540. Thus, the fluorescent light 549 emitted toward the detector is sufficiently stable and uniform to provide a true measurement of the measurement region 591.

Thus, although measurements are made with a UV emitter at about 15cm, measurements using only visible light emitters, for example for measuring anthocyanins, can be performed at greater distances, even up to about 1 m.

In other embodiments, the beam of the transmitter may be oriented parallel to the detection axis, for example for large distance measurement applications.

Fig. 7 shows the structure of the device in this embodiment of the invention in more detail. The cylindrical part 513 of the cartridge contains a substantially annular electronic card 5131 comprising the power supply circuit of the excitation source, the management circuit of the excitation source and the circuit of the generation current generator.

The probes and accompanying electronics are grouped together in a cylindrical probe module 5400, placed at the center of the circle formed by the excitation emitter on the measurement surface 514, and extending in the direction of the object to be analyzed. The outer surface of the cylinder carries three

detectors

541, 542 and 543.

This arrangement makes it possible to place the probe at substantially the same level as the end of the emitter, so that it has a wide reception range and is thus accessible to the target.

The detection module 5400 comprises three small

electronic cards

5141, 5142 and 5143, substantially similar, substantially circular and stacked along their longitudinal axis, fixed and separated by small columns 5144.

The first small electronic card 5141 is located on the outer surface side of the detection module 5400, carrying the detection elements (here silicon photodiodes). For each detector, the silicon photodiode 5420 receives light to be detected through a color or high pass filter 5421 and an interference filter 5422, which are removably secured by a retaining nut that is seated in a 25.4mm cylindrical opening and thereby can accommodate a standard 1 inch or 25 mm filter.

The second small electronic card 5142, removed from the measuring surface 514, carries the circuit and amplifier to cancel the ambient light through a negative feedback loop.

The third small electronic card 5143 carries the circuits and components contained in particular in the track and hold unit.

The detection module 5400 constitutes a compact assembly that is removable from the cartridge 510, e.g., for maintenance, or is replaced by a camera module or a module that includes one or more optical waveguides.

The detection module is connected to a processing module 5151 through an opening in the large electronic card 5131, the processing module 5151 being located on the interface side and comprising all or part of the processing means, in particular the acquisition unit and the computing means.

The process module is included in a part 515 that carries the screen 5152 on the cartridge, which part can be tilted for ease of reading and can be retracted into the compartment 5105 in the cartridge 510. The processing module may include a detachable connection to allow it to be easily replaced, for example, for updating or changing functions.

Thus, the electronics 5131 connected to the excitation, the electronics 5141 to 5143 connected to the detector, and the processing module 5151 are provided in separate and distinct electronic modules, allowing for simplified maintenance. The modules are also spaced apart by a distance, here 2cm and for example at least 1.5cm, allowing better heat dissipation and limiting the risk of interference between the circuits included in the modules.

In the embodiments described herein, the detection is synchronized with the excitation emitted by the excitation emitter. Excitation at a frequency of 1kHz with 520 millisecond pulses has been used successfully and allows real time processing and coverage of the site when needed. During the measurement cycle, the different fluorescence measurements required to establish the content or programmed index are alternated.

Thus, the management and processing means are arranged to:

-transmitting a control signal for controlling the transmitter by means of a pulse;

-detecting the fluorescent peaks generated by these pulses, the amplification within the detector being such that ambient light is rejected by a negative feedback loop;

-controlling the processing of the detected fluorescence peak, for example by a track and hold unit, by for example the same control signal; and

-providing an analog measurement of the fluorescence measurement to the acquisition unit.

In other embodiments, synchronous detection is provided using phase modulation between excitation and detection. In this way, the management and processing unit is arranged to:

-controlling the transmitter according to a frequency comprising phase modulation;

-processing the fluorescence detection signals in phase demodulation and providing fluorescence measurements.

The determination of the Anthocyanin (ANTH) content in grape peel will now be described at variable distances according to the first aspect of the invention.

Fig. 8 shows the absorption spectrum 82 of malvidin (the predominant anthocyanin in grapes) in 50% acidified methanol, the emission spectrum 83 of chlorophyll, and the transmission spectrum 84 of filter F2(Schott RG9 filter) in extinction coefficients.

The source 11 is a source emitting GREEN (GREEN) radiation 111, with a wavelength within the absorption band of anthocyanins, which initiates fluorescence of chlorophyll 33 and causes emission of fluorescent radiation 112. The wavelength of the source 111 may be, for example, about 530 nm. Fig. 8 shows a spectrum 86 of the radiation 11. An example of a GREEN source is the NS530L diode of Roithner Lasertechnik.

The source 12 emits RED (RED) radiation 121, slightly or not at all absorbed by the anthocyanin, which initiates fluorescence of chlorophyll 33 and causes emission of fluorescent radiation 122, which serves as a reference for measuring anthocyanin content. The wavelength of this source is preferably 650 nm. Fig. 8 shows a spectrum 85 of radiation 121.

Furthermore, the source 13 emits BLUE (BLUE) radiation 131, slightly or not absorbed at all by the anthocyanin, which triggers the fluorescence of chlorophyll 33 and causes the emission of fluorescent radiation 132, which serves as a supplementary reference for measuring anthocyanin content. The wavelength of this source is preferably 450 nm. Fig. 8 shows a spectrum 87 of the radiation 12.

Sources

11, 12 and 13 successively irradiate tissue 30 by emitting

radiation

111, 121 and 131, respectively. Depending on the anthocyanin content 34, the radiation 111 is absorbed by the epidermis of the pericarp in variable amounts, whereas the red 121 and blue 131 radiation is not or only slightly absorbed. The

radiation

111, 121 and 131 all induce fluorescence of chlorophyll 33 and then emit red or near infrared

fluorescent radiation

112, 122 and 132, respectively, with an intensity proportional to the intensity of the

radiation

111, 121 and 131 received by chlorophyll. The content of anthocyanin in the grape peel can be determined by measuring the ratio of the fluorescence emissions excited by source 11 and source 12. Furthermore, measuring the ratio of the fluorescence emissions excited by source 12 and source 13 can determine whether reference radiation 12 is affected by an increasing amount of anthocyanin over time. As the anthocyanin content of grapes increases, radiation 111 is excessively absorbed and begins to saturate. In addition, anthocyanins also begin to affect reference radiation. In this case, in order to perform measurement under the super-saturation caused by excessively high concentration of anthocyanin, the radiation 121 as the reference radiation before saturation becomes the measurement radiation after saturation, and the radiation 131 as the supplementary radiation before saturation becomes the reference radiation after saturation. The radiation 111 is no longer used for measurement. This allows to perform anthocyanin content measurements in supersaturated conditions.

Then, the anthocyanin content was calculated according to the following expression:

ANTH_{before saturation}＝logFER_ANTH_GREEN-logFER_CHL_G

ANTH_{After saturation}＝C+logFER_ANTH_RED-logFER_CHL_R

Wherein,

1.logFER_ANTH_GREEN＝log(FRF_{red wine}/FRF_Green)

2.logFER_ANTH_RED＝log(FRF_{Blue (B)}/FRF_{Red wine})

3.logFER_CHL_G＝log(FRF_{Red wine}/FRF_Green)

4.logFER_CHL_R＝log(FRF_{Blue (B)}/FRF_{Red wine})

In these expressions, logFER _ CHL_G＝log(FRF_{Red wine}/FRF_Green)，logFER_CHL_R＝log(FRF_{Blue (B)}/FRF_{Red wine})，logFER_CHL_GAnd logFER _ CHL_RThe amount is measured before anthocyanin appears in grape skin, and the ANTH is the anthocyanin content in grape peel, FRF, in nanomoles per square centimeter_Green、FRF_{Red wine}And FRF_{Blue (B)}The intensity of the

fluorescence radiation

112, 122 and 132 respectively induced by the

radiation

111, 121 and 131, C being such that ANTH occurs at the instant of saturation_{After saturation}Equal to ANTH_{Before saturation}Is constant.

Fig. 9 shows a schematic representation of the development of the measured anthocyanin content in grape peel, determined according to the first aspect of the invention. Axis 91 represents the time at which saturation compensation begins to be performed. Curve 92 shows the result obtained with the saturation compensation according to the invention, i.e. with the reference radiation before saturation as measurement radiation after saturation and the supplementary radiation before saturation as reference radiation after saturation. Curve 93 shows the result obtained without performing saturation compensation. It should therefore be noted that, without carrying out a saturation compensation, the curve 93 obtained decreases after saturation, while the anthocyanin content continues to increase. By compensation, an increasing curve 92 is actually obtained, indicating an increase in anthocyanin content in the grape peel.

The determination of the anthocyanin content in grape peel at variable distances according to the second aspect of the invention will now be described. According to a second aspect, the wavelength of the radiation 121 remains unchanged at 650 nm. On the other hand, the wavelength of the radiation 111 is chosen to be 590 nm. This wavelength corresponds to a lower absorption of the anthocyanin compared to the wavelength 530nm selected according to the first aspect of the invention, which corresponds to an absorption peak of the anthocyanin. Fig. 10 shows the absorption spectrum 82 of malvidin (the desired determined amount of anthocyanin) in 50% acidified methanol used in the second aspect of the invention, the emission spectrum 83 of chlorophyll, and the transmission spectrum 84 of filter F2, the spectrum 85 of radiation 121, and the spectrum 88 of radiation 111.

The measurement principle of the second aspect of the invention is substantially similar to that of the first aspect of the invention. The

sources

11 and 12 successively irradiate the tissue 30 by emitting

radiation

111 and 121, respectively. Depending on the anthocyanin content 34, radiation 111 is absorbed by the epidermis of the pericarp in variable amounts, while red radiation 121 is not absorbed.

Radiation

111 and 121 induce fluorescence of chlorophyll 33, which then emits measured

fluorescent radiation

112 and 122, respectively. However, since the absorption corresponding to the wavelength of 590nm chosen for radiation 121 is lower than the absorption corresponding to the wavelength of 530nm chosen for radiation 121 in the first aspect of the invention described above, there is no saturation due to the increased anthocyanin content in the grape pericarp. Fig. 11 shows:

● first curve 1111 which shows the variation of the measurement performed with radiation 121 having a wavelength of 530nm as a function of the anthocyanin content in the grape peel; and

● second curve 1112 showing the measurement performed with radiation 121 having a wavelength of 590nm as a function of anthocyanin content in the grape peel.

The wavelength 590nm corresponds to a lower absorption of anthocyanin than the wavelength 530 nm. It should be noted that for the wavelength of 530nm (curve 1111), saturation is reached very quickly, whereas for the wavelength of 590nm (curve 1112), no saturation occurs. Furthermore, the results obtained in the case of curve 1112 are more linear than those obtained in the case of curve 1111.

Advantageously, the first and second aspects of the invention may be combined. In fact, the supplemental radiation 131 described above can be used to determine whether the reference radiation 121 is affected by anthocyanins. When the concentration or content of the anthocyanin is such that saturation occurs even in the case where the wavelength of the radiation 111 shifts relative to the absorption peak (e.g., 590nm), the radiation 121 that was the reference radiation before saturation may be taken as the measurement radiation after saturation, and the radiation 131 that was the supplemental radiation before saturation may be taken as the reference radiation after saturation. Figure 12 shows the results obtained by combining the first and second aspects of the invention. Curve 1201 corresponds to the results of measurements with the following radiation:

-radiation 111 at 530nm,

radiation 121 at 650nm, and

-radiation 131 at 450 nm;

curve 1201 corresponds to the results of measurements with the following radiation:

-radiation 111 at 590nm,

radiation 121 at 630nm, and

radiation 131 at 450 nm.

Axis 1203 represents the time at which saturation compensation begins to be performed according to the first aspect of the present invention.

Furthermore, the wavelengths of

radiation

111, 121, and 131 may be modified according to changes in the pH of the biological tissue that it is desired to characterize. Fig. 13 shows an absorption spectrum 1301 of anthocyanin at pH 1 and an absorption spectrum 1302 of anthocyanin at pH 8. It should be noted that as the pH increases, the absorption peak shifts to higher wavelengths. Such variations in the absorption spectrum can be taken into account in the saturation compensation, regardless of which aspect of the invention is implemented.

The measurement of Flavonol (FLAV) content in grape skins according to the third aspect of the invention will now be briefly described. In this case, source 11 is a source emitting Ultraviolet (UV) radiation 111, the wavelength of which is within the absorption band of flavonol, which induces fluorescence of chlorophyll 33 and causes emission of fluorescent radiation 112. The wavelength of the source 11 may be, for example, about 375 nm. The source 12 may emit reference radiation 121 at 650nm, which corresponds to a wavelength that is not absorbed by flavonol. When the measuring radiation 111 is saturated by the flavonol content in the peel, a measuring radiation of 450nm corresponding to the lower absorption in the flavonol absorption spectrum can be used. This radiation of 450nm may be emitted by the source 13, for example. The reference radiation is not modified but remains at 650 nm.

The invention can also be used to measure the content of other compounds that are chromophoric and have a slight or no fluorescence, for example lycopene in tomatoes.

The present invention is not limited to the above-described embodiments and may be used to characterize any type of biological tissue.

Claims

1. Method for determining the content of a non-fluorescent chromophoric compound (34), referred to as first compound, in a biological tissue (30) of a biological entity, the biological tissue (30) further comprising a fluorescent chromophoric compound (33), referred to as second compound, the method comprising at least one iteration of the following operations:

-emitting, by emitting means (11, 12), a first radiation (111), called measurement optical radiation, and a second radiation (121), called reference optical radiation, in the direction of the biological tissue (30), each of the first and second radiation (111, 121) being selected to generate a fluorescent radiation of the second compound (33), each of the first and second radiation (111, 121) being partially absorbed by the first compound (34);

-measuring the fluorescence radiation (112, 122) induced by the first and second radiation (111, 121) by a measuring device (21, 22); and

-determining the content of the first compound (34) in the biological tissue (30) from the measurement of the measurement device,

characterized in that the method further comprises at least one compensation of the measurement saturation, which is due to too high absorption of the measurement optical radiation (111) by the first compound (34), the compensation comprising: selecting a wavelength for the measurement optical radiation that corresponds to a lower absorption in the absorption spectrum of the first compound (34).

2. The method according to claim 1, further comprising emitting, by an emitting device (13), a third radiation (131), called complementary optical radiation, in the direction of the biological tissue (30), the third radiation being selected to cause a fluorescent radiation (132) of the second compound (33), the method further comprising measuring the fluorescent radiation (132) caused by the complementary optical radiation (131), the measurement being used to determine whether the reference optical radiation (121) is affected by the first compound (34).

3. The method according to claim 2, characterized in that, when the content of the biological tissue (30) in the first compound (34) affects the reference optical radiation (121), the wavelength selected for the measurement optical radiation (111) after saturation corresponds to a wavelength around the wavelength of the reference optical radiation (121) before saturation.

4. The method of claim 3, further comprising: when the distance between the biological tissue (30) and the emitting means (11, 12, 13) and the measuring means (21) is not fixed, a new wavelength is selected for the reference optical radiation after saturation, which is less affected by the content of the first compound.

5. The method according to claim 4, characterized in that the new wavelength selected for the reference optical radiation after saturation corresponds to a wavelength around the wavelength of the supplementary optical radiation (131) when the distance between the biological tissue (30) and the emitting means (11, 12, 13) and the measuring means (21) is not fixed.

6. The method according to any one of the preceding claims, wherein the wavelength selected for the measurement optical radiation (111) is less absorbing than a limiting wavelength for which a potential saturation would occur at a desired maximum content of the first compound (34) in the biological tissue (30) when the reference wavelength (121) is not influenced by the content of the first compound (34) in the biological tissue (30) corresponding to the first compound (34).

7. The method of claim 1 or 2, further comprising: -modifying the wavelength of the measurement optical radiation (111) and/or the reference optical radiation (121) according to a physicochemical characteristic having an effect on the absorption spectrum of the first compound (34) and/or on the fluorescence spectrum of the second compound (33).

8. The method according to claim 1 or 2, wherein at least one of the reference optical radiation (121) and the measurement optical radiation (111) is emitted with a predetermined intensity and the other radiation is emitted with a variable intensity, such that the fluorescence radiation induced by each of the reference optical radiation (121) and the measurement optical radiation (111) has an equal intensity.

9. The method of claim 8, wherein the intensity of the measurement optical radiation is adjusted in accordance with a control signal related to fluorescent radiation induced by the measurement optical radiation and the reference optical radiation.

10. The method according to claim 1 or 2, characterized in that the intensities of the measurement optical radiation (111) and the reference optical radiation (121) are alternately varied by a phase shift at a predetermined frequency.

11. The method of claim 2, further comprising synchronizing the first, second and third radiations (111, 121, 131) by phase modulation.

12. The method according to claim 1 or 2, characterized in that each of the measurement optical radiation (111) and the reference optical radiation (121) is emitted in pulses.

13. The method according to claim 1 or 2, wherein determining the content of the first compound (34) comprises calculating the ratio:

fluorescence induced by the reference optical radiation (121)

Fluorescence induced by the measurement optical radiation (111).

14. The method of claim 1 or 2, further comprising: determining the development of the content of the first compound (34) in the biological tissue (30) over time.

15. The method of claim 2, wherein the biological tissue is grape peel and the second compound is chlorophyll;

-when the first compound is an anthocyanin,

-the wavelength of the measuring optical radiation before saturation is between 500 and 600 nm;

-the wavelength of the reference optical radiation before saturation is between 600 and 700 nm;

-the wavelength of the supplementary optical radiation is between 400 and 500 nm;

-the wavelength of the measuring optical radiation after saturation is between 600 and 700 nm;

-the wavelength of the reference optical radiation after saturation is between 400 and 500 nm; and

-when the first compound is a flavonol,

-the wavelength of the measurement optical radiation before saturation is between 300 and 400 nm;

-the wavelength of the measuring optical radiation after saturation is between 400 and 500 nm;

-the wavelength of said reference optical radiation after saturation is between 600 and 700 nm.